Both non-pathogenic and pathogenic Burkholderia, including members of the opportunistic infection-causing Burkholderia cepacia complex, encode genes to produce contact-dependent growth inhibition (CDI) system proteins. CDI is a widespread phenomenon in which Gram-negative bacteria use the toxic C-terminus of a polymorphic surface-exposed exoprotein to inhibit the growth of susceptible bacteria upon cell-cell contact. The exoprotein toxin is delivered to the cytoplasm of target cells and autoinhibition is prevented by production of a small immunity protein. We previously demonstrated that, in addition to their role in interbacterial competition, the CDI system proteins in Burkholderia thailandensis, encoded by bcpAIOB, mediate biofilm formation. This requires the putative nuclease activity of the BcpA toxic exoprotein, but is independent of interbacterial killing, suggesting that biofilm formation in B. thailandensis involves an uncharacterized function of BcpA. Preliminary data suggest that bcpAIOB mediate interbacterial signaling in B. thailandensis, leading to a change in gene expression in immune target bacteria. Given these data and the conservation of CDI systems, it seems likely that CDI systems in other Burkholderia also function in interbacterial signaling and could play a role during infection. Toward understanding the role of CDI system-mediated signaling, this proposal will test the hypothesis that BcpA toxin delivered to an immune target cell interacts with specific periplasmic and cytoplasmic factors, leading to global gene expression changes, and represents a useful therapeutic target in pathogenic Burkholderia. Experiments proposed in Aim 1 will continue to investigate the mechanism of bcpAIOB-mediated interbacterial signaling in B. thailandensis, defining the affected gene regulation pathways and identifying required regions of BcpA, as well as other factors necessary for signaling. Studies in Aim 2 will explore the role of CDI systems and their potential for exploitation as therapies in pathogenic Burkholderia cepacia complex strains. These studies represent the first investigation of what may be a widespread phenomenon in Gram-negative bacteria and will work toward addressing an essential need for new drug targets and antimicrobials.